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September 2, 2005September 2, 2005

Integrated Micro/Nano Summer Undergraduate Research Experience

Combinatorial Bioassays in Droplet Arrays for Combinatorial Bioassays in Droplet Arrays for Monitoring Astronaut Health During Space TravelMonitoring Astronaut Health During Space Travel

Liana Alston, Department of Biochemistry, UC Riverside

Faculty Mentor:Dr. Abraham P. Lee, Department of Biomedical Engineering, UC Irvine

Graduate Mentor:Tsung-Hsi Albert Hsieh, Department of Biomedical Engineering, UC Irvine

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Background & TerminologyBackground & Terminology

• Microfluidics is a multidisciplinary field comprising physics, chemistry, engineering and biotechnology that studies the behavior of fluids at the microscale

• Polydimethylsiloxane (PDMS) is the most widely used silicon-based organic polymer, and is particularly known for its unusual flow properties. It is optically clear, and is generally considered to be inert, non-toxic and non-flammable.

Phloem:The tree equivalent to

veins of the human body. Essentially it is a system of tubes that transport sugar and

other organic nutrients throughout

the plant.

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Who, What & Where:Who, What & Where:Goal of this ProjectGoal of this Project

The development of a new diagnostic tool, that will require only a small salivary sample to perform rapid DNA analysis with a molecular beacon detector, resulting in an immediate qualitative verification of viral infection.

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Why:Why:Potential Applications of this ResearchPotential Applications of this Research

• Non-invasive diagnosis of astronauts with reactivated and potentially symptomatic herpesvirus infection

• Non-invasive diagnosis of individuals with viral infections, in a domestic setting, to perhaps stop the spread of pandemic diseases

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How:How:Project OutlineProject Outline

I. Literature Searches for a Virus to Attempt to Detect in Astronaut Saliva

II. Hybridization Buffer Optimization

III. Microchannel DesignIV. Microchannel FabricationV. Droplet Generation Experiments

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Journal & Review Paper Search Journal & Review Paper Search Protocol & ResultsProtocol & Results

• Criteria…a VIRUS that can be diagnosed via a salivary sample…a VIRUS that can be detected by DNA/RNA sequence in saliva…a VIRUS with a high accuracy (~80%+)

sensitivity: # ill individuals correctly diagnosedspecificity: # well individuals (% of control group) in which virus

was not detected

• Most Convincing Papers

7 Y1997article37%gene (DNA)/ PCRHHV 8

Y1998article70%gene (RNA)/ PCRAdult Periodontal Disease

gene (DNA)/ PCR

N1994article71%gene (DNA)/ PCRHead & Neck Cancer

N1996article84%gene (DNA)/ PCRGastric H. Pylori Infection

N1994article48%gene (RNA)/ PCRHepatitis C

N1999article90%HHV-6 & 7

Y1996article95%100%elevated [creatinine] Renal Disease

Y1985article97%75%gene (DNA)/ PCREBV

SpecificitySensitivityCould Affect an

AstronautYear

Link to PDF file

Accuracy Method of Detection Disease

Y2002article72%gene (DNA)/ PCRVaricella-Zoster Virus

PBD

Y199081%anitbody (CA 125)Ovarian Cancer

HSV-1

EBV

Dengue

Parvovirus B 19

Gastric H. Pylori Infection

HIV

Neurocysticercosis

Rubella

Mumps

Measles

Hepatitis C

Hepatitis B

Hepatitis A

Y1986article97%55%antibody (IgG) / ELISA

article88%

Y1993article82%antibody

Y2005article90%antibody

N 1998article100%92%antibody

N1996article95%100%antibody

Y1997article94%97%antibody

N1997article97%97%antibody

N 1990article100%antibody

N"article98%98%antibody (IgG & IgM)

N "article94%94%antibody (IgG & IgM)

N 1994article100%97%antibody (IgG & IgM)

N"article100%100%antibody

N "article100%100%antibody

Y1992article98%100%antibody (IgG & IgM)

PBD

Y199081%antibody (CA 125)Ovarian Cancer

HSV-1

EBV

Dengue

Parvovirus B 19

Gastric H. Pylori Infection

HIV

Neurocysticercosis

Rubella

Mumps

Measles

Hepatitis C

Hepatitis B

Hepatitis A

Y1997article61%gene (DNA)/ PCRCMV

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Contribution to Monthly NASA ReportContribution to Monthly NASA Report

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Buffer Characterization:Buffer Characterization:an Overviewan Overview

• Point: Maximize Signal to Background Ratio (SNR) of the MB-cDNA hybridization

• Varied [MgCl2], pH, & [KCl] in that order– Ranges:

• 0,1,5,10,20,50,100 mM MgCl2 concentrations• pH 7, 8, 8.5, 9 • 0, 10, 50, 100 mM KCl concentrations

• Smaller vs. Higher Concentrations of MB and cDNA-expensive!

• Our best buffer vs. IDT’s working buffer• Hepatitis C vs. Breast Cancer MB & cDNA• Albert’s cDNA vs. IDT’s cDNA

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Buffer CharacterizationBuffer Characterization

• Noteworthy EquipmentNoteworthy Equipment-Spectrofluorometer-RT-PCR machine-Autoclave -Automatic Delivery pipets-Eppendorf Tubes-Black Microwell Plates-pH meter

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Buffer CharacterizationBuffer Characterization

STNR as a function of MgCl2 after denaturing/cooling of sample

020000400006000080000

100000120000140000

1 10 50

[MgCl2]

Fluo

resc

ence

noise

signal

STNR as a function of [MgCl2] Hepatitis C

0

5000

10000

15000

0 0 1 5 5 10 20 20 50 100 100

[MgCl2]

Fluo

resc

ence

before

after

STNR as a function of pHHepatitis C

0100020003000400050006000700080009000

10000

7 7 7 8 8 8 8.5 8.5 8.5 9 9 9

pH

Fluo

resc

ence

before

after

STNR as a function of [KCl]Hepatitis C

0

2000

4000

6000

8000

10000

0 0 0 10 10 10 50 50 50 100 100 100

[KCl]

Fluo

resc

ence

noise

signal

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• Trends observed in Hepatitis C: – 50mM = optimal [MgCl2] 0-100mM– pH 7 = optimal pH 7,8,8.5,9– 50mM = optimal [KCl] 0-100mM…w/ both small (0.05: 0.25) and high (0.5:2.5mM)

MB:cDNA concentrations.

Buffer CharacterizationBuffer Characterization

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PDMS Microchannel FabricationPDMS Microchannel Fabrication

• Pour PDMS• Vacuum to eliminate air bubbles• Bake• Peel off PDMS channel; cut & punch

holes• Use N2 gun to blast away PDMS

crumbs to avoid intra-channel blockage

• O2 plasma (CH3 -> OH) render glass slide and PDMS hydrophobic, so droplets won’t stick to channel

• Seal PDMS channel to glass slide

• Si wafer• Spin coat SU-8• Bake• Photoresist• Expose• Post-expose bake• Develop• Rinse and Dry

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Mask Design; Droplet Generation Mask Design; Droplet Generation ExperimentExperiment

Digital Syringe Pumps

L-edit rendition of µchannel incorporating Tim’s “Fusion Turns”

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Droplet Generation ExperimentDroplet Generation Experiment

Tim’s Experiment:

H20 and H20

Liana & George’s Experiment:

H20 and dye

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Future WorkFuture Work• Optimize flow rates for fusion turn

channels – H20 + dye

• Using Metamorph to analyze the mixing within droplets in 0 vs. 90 vs. 180 degree channels

• Continue buffer characterization for more conclusive data supporting an “optimal” working buffer (esp. important for detecting reactivation of a latent virus

• Finding unique EBV sequence – Unique to EBV (relative to

other gammaherpesvirinae– Unique to reactivated EBV

relative to latent EBV

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AcknowledgementsAcknowledgements Graduate Students

Albert Tsung-Hsi Hsieh Tim Wei-Yu Tseng Jason Lung-Hsin Hung Jeff Fisher Joe Harris & Grace, Dr. Jim Brody’s lab group Wajeeh, Dr. Noo Li Jeon’s lab group

Undergraduate Students Adam Yuh Lin Patrick Pan George Yung-Chieh Chen Alok Vij

UROP Staff Said Shokair Jerry McMillan

INRF Staff Goran Matijasevic

Arroyo Vista IM-SURE house

IM-SURE voyage